Abstract

Nearly 200,000 individuals worldwide are diagnosed with a form of soft-tissue sarcoma (STS) every year. Due to a lack of novel targeted therapeutics sarcoma for these malignancies treatment has not changed significantly in 25 years. Therefore, the discovery of novel targets and mechanisms is critical. One promising avenue the Hippo pathway, which we have identified as a key regulator of sarcomagenesis in undifferentiated pleomorphic sarcoma (UPS), a commonly diagnosed and aggressive subtype of sarcomas. Inactivation of the pathway, promotes nuclear localization of YAP1, a transcriptional co-activator that promotes proliferation. Whereas, many studies have defined YAP1’s transcriptional targets in epithelial tumors and normal tissues, its role in mesenchymal tumors is unclear. Here, we confirm that increased YAP1 mRNA expression correlates with worse overall survival in UPS patients. To define mechanisms of YAP1-mediated sarcomagenesis, we developed a novel genetic mouse model in which YAP1 is conditionally deleted in UPS tumors. Microarray analysis of these tumors revealed that YAP1 loss inhibits expression of NF-κB targets. We have performed ChIP-seq and super-enhancer analysis of human UPS tumors and found that many of the 900 identified super enhancers regulate expression of NF-κB targets that are dependent on YAP1 in our system. This finding is particularly relevant as NF-κB is a key regulator of muscle cell proliferation and suppresses myoblast differentiation. Together, these data suggest that upregulation of YAP1 activity promotes NF-κB-mediated proliferation and inhibits differentiation, resulting in sarcomagenesis. Importantly, we have discovered that a combination of JQ1, a BET family inhibitor, and SAHA, a histone deacetylase inhibitor, decreases YAP1 expression, YAP1 protein stability, and sarcoma cell proliferation. SAHA/JQ1 treatment significantly increased expression of Angiomotin (AMOT), which binds YAP1, sequesters it in the cytoplasm, and facilitates it degradation. We have also found that AMOT expression is lost in sarcomas, likely due to epigenetic suppression. Consistent with these observations, SAHA/JQ1 treatment also decreased gene expression of YAP1 targets and caused re-expression of the muscle differentiation markers p57, MEF2C, and MYOD1. We have investigated the effect of of SAHA/JQ1 in vivo and found dramatic inhibition of tumor growth, as well as reduced YAP1 expression, and increased AMOT expression. Importantly SAHA/JQ1 treatment also inhibited NF-κB activity. Our studies have revealed for the first time that YAP1 expression is epigenetically modulated through AMOT de-regulation in sarcomas, resulting in elevated NF-κB activity and sarcomagenesis. SAHA/JQ1 treatment re-establishes epigenetic control of the Hippo pathway reducing proliferation and enhancing differentiation. Ultimately, we will determine whether this approach represents a novel course of targeted treatment for sarcoma patients.